The field of the invention is the fermentation of biomass substrates with thermophilic anaerobes to produce alcohols, especially ethanol. More particularly, the invention is concerned with novel mutants of Clostridium thermosaccharolyticum which are capable of producing higher yields of ethanol.
Lee and Ordal.sup.1, in 1967, reported that C. thermosaccharolyticum produced ethanol as one soluble end product of the dead cells of a fermentation by glucose-grown cells in Warburg flasks at 56.degree. C. More recently, Ljungdahl and Wiegel.sup.2 have reported on ethanol fermentations using strains of anaerobic thermophilic bacteria at the 27th IUPAC Congress in Helsinki, 1979. Some of the strains were identified as Clostridium thermohydrosulfuricum, previously isolated only from extraction juices obtained in an Austrian sugar beet factory. A second strain of anaerobic thermophilic bacterium was also identified with a proposed name of Thermoanaerobium ethanolicum. This bacterium has a temperature range of 37.degree. to 78.degree. C. and was isolated from the mud samples taken from hot springs. It differs from C. thermohydrosulfuricum in that it does not form spores and produces, during fermentations, almost 2 moles of ethanol and carbon dioxide per mol of fermented glucose with lactate, acetate, and H.sub.2 as minor products.
A type of vacuum fermentation system, including a cell recycling procedure, for the production of ethanol has been described by Cysewzki and Wilke.sup.3. Their studies were conducted using a yeast, Saccharomyces cerevisiae (ATCC No. 4126), at a fermentation temperature of 35.degree. C. A 10% glucose feed was employed and a cell density of 50 g dry wt/liter was obtained in an atmospheric-cell recycle fermentation which produced a fermentor ethanol productivity of 29.0 g/liter-hr. The vacuum fermentor eliminated ethanol inhibition by boiling away ethanol from the fermenting beer as it was formed. At a total pressure of 50 mm Hg using a 33.4% glucose feed, ethanol productivities of 82 and 40 g/liter-hr were achieved with the vacuum system with and without cell recycle, respectively. Fermentor ethanol productivities were thus increased as much as twelvefold over conventional continuous fermentation. However, in order to maintain a viable yeast culture in the vacuum fermentor, a bleed of fermented broth had to be continuously withdrawn to remove nonvolatile inhibiting compounds. It was also found necessary to sparge the vacuum fermentor with pure oxygen periodically to satisfy the trace oxygen requirement of the fermenting yeast.
Dr. Edward J. Hsu, (the inventor named herein) and his associates have been working with Clostridium thermosaccarolyticum (National Canners Association strain 3814).sup.4. This work stemed from the initial observation that this species of Clostridia is highly resistant to heat at normal sterilization temperatures. Lee and Ordal.sup.1 earlier had found ethanol as an end product from the fermentation of such cells.
In this work leading up to the present invention, involved other developments relating to the use of anaerobic thermophiles in ethanol production. These developments are described below in abbreviated form:
(1) One development was the continuous cultivation of the oxygen-sensitive clostridia to induce a morphological change such as elongation and/or sporulation..sup.4,5
(2) A further development was the finding that metabolic change occured as morphological changes proceeded..sup.6 Little or no ethanol was apparent, until the cells elongated and developed into sporangia.
(3) Still another important finding was that of the conversion of acetate to ethanol in cells undergoing elongation and sporulation..sup.6,7 De-repressed levels (78 times) of ethanol dehydrogenase, and higher levels of NADPH were also found in the elongating cells.
(4) It has also been determined that elongating cells producing predominate amounts of ethanol and dehydrogenase are essentially non-dividing cells. These cells can be arrested at this stage, so that sporulation does not proceed;.sup.7 in fact, highly synchronized cultures can be obtained by an interruption in cell division, and sporogenesis..sup.8 This last discovery implies that sporulation per se is not essential to ethanol production, and that the change in cell length is perhaps responsible for the metabolic shift.
Each of these four developments described above had disadvantages and were found insufficient to establish a viable ethanol production system. It was found that the continuous culture to induce morphological change required that the organism be cultivated at an extremely low concentration (density), therefore, relatively low concentration of ethanol was produced.
There were also some disadvantages found in the second development relating to the shifting metabolism, these were: (1) little or no ethanol was produced by the short cells; (2) the amount of ethanol produced became significant only after the majority of cells started to elongate; and (3) the amount of ethanol became predominate only for a very short period of time, i.e. only by cells that were successfully converting to sporangia.
In the third development above, the cultivation technique to de-repress levels of ethanol dehydrogenase, it was found that the ethanol concentration was dependent on synchrony of the population, which was difficult to maintain.
In the fourth development, a disadvantage observed was that the method was mostly unsuccessful due to unstable physiological conditions. Harvesting such an organism would be extremely difficult and probably not economically feasible.